Buffy coat / Microhematocrit technique
- why use upconcentration by centrifugaton, followed by light and fluorescent microscope examination, using more staining techniques for visualizing microbes?

By Marie Kroun, MD (MK), 2012

With use of the microhematocrit or buffy-coat technique, i.e. by centrifugation of the anticoagulated full blood sample, it is wellknown from articles dating back to the 1970ies (see references below), that free microbes in the plasma, like borrelia, trypanosoma, microfilaria, other? .. will accumulate in the platelet rich plasma fraction, just above the white buffy-coat layer (layer of white blood cells, i.e. immune cells), while the red blood cells with intracellular organisms inside, like malaria or babesia, will accumulate in the layer of red blood cells just below the  buffy-coat layer:
 
centrifuged blood, buffy-coat by Marie Kroun, MD         QBC
After one centrifugation (picture by MK); the picture on the right is borrowed from: http://www.malariasite.com/malaria/QBC.htm 

After centrifugation of an anticoagulated (EDTA or citrate) whole blood sample, wet drops (1 blood drop ~ 50 microliter; calculated from 20 drops per ml) can be examined directly, unstained or stained with immunofluorescent stain, under a cover glass, or can be smeared onto an object glass, dried and stained as described below.
F
rom the around 0,5 ml of buffy-coat fraction gained per 5 ml blood sample, can be made 10-20 bloodsmears, and any residual material is frozen in the syringe, could perhaps be used for confirming PCR test, if sign of microbes are found by the microscopy? - see video on how buffy-coat smear preparation is made. Dried blood smears has a very long durability (20+ years) when stacked closely together well protected. I cover the upper smear in a stack with a clean object glass with ID date on a label, and fix the stack closely together with tape. If not stored properly the dried blood may be accessible to insects i.e. "silverfish" (Lepisma saccharina) that like to eat it. After drying, the blood smears can be fixed and stained by any conventional hematology blood stain, or a Romanovsky stain type, like Diff-Quik: http://en.wikipedia.org/wiki/Diff-Quick (which is easy and fast) or preferably by a microbe specific immunestain method, if one has access and money to buy relevant target specific antibodies, or the detection of DNA/RNA can be enhanced by staining with acridine orange and by using a fluorescence microscope...

Acridine orange: http://en.wikipedia.org/wiki/Acridine_orange
BD reagent droppers (Eng.): http://www.bd.com/ds/productCenter/261182.asp 
(Dansk: http://www.bd.com/resource.aspx?IDX=19360)
QUOTE: "Acridine orange binds to nucleic acids of cells and bacteria [DNA, RNA]. When viewed under UV light, single-stranded DNA and RNA fluoresce orange, whereas double-stranded DNA appears green. At low pH (3.5 – 4.0), bacteria and fungi stain bright orange.
Cellular material stains pale green to yellow.2   Nuclei of activated leukocytes may stain orange, yellow or red, depending upon the degree of increased RNA production.
Erythrocytes either have no color or appear pale green."

Acridine Orange for malaria diagnosis: its diagnostic performance, its promotion and implementation in Tanzania, and the implications for malaria control (2002)
http://www.cytoscience.com/acridine%20orange%20test%20keiser%20et%20al.pdf
"The literature on the discovery, development and validation of the AO [acridine orange] method for malaria diagnosis is reviewed here."
"The basic feature of fluorescent staining is that, in contrast to the mature erythrocytes that do not contain DNA or RNA, the nucleic acids of the parasite fluoresce strongly."
"After testing 30 different fluorescent dyes, Fuhrmann (1962) found an AO solution with a pH between 6 and 7 and a concentration of 1: 10,000 to be the most promising dye for the staining of thin bloodsmears containing Babesia canis, P. berghei or P. cathemerium. ... shortly thereafter, excellent results were reported for the AO staining not only of thin smears containing P. berghei or P. vivax (Ambroise-Thomas et al., 1965) but also of thick smears containing various Plasmodium species (Sodeman, 1970). In the latter study, the thick smear was made slightly thinner than was then customary and dehaemoglobinized during staining with a 0.01% AO solution of pH 5.4 (Sodeman, 1970). Commonly, AO stain is applied to a thin smear only after the smear has been dried and then fixed in methanol. However, an immediate method, in which AO solution is applied directly to an unfixed and undried blood film, has also been described (Kawamoto, 1991 a; Metzger and Nkeyi 1995)".
Fluorecens microscopy was for a long time too expensive but ... "In 1991, however, the development of a relatively cheap, interference-filter system (a multi-layered excitation filter combined with a barrier filter) designed for use with AO in a standard light microscope provided a low-cost tool for malaria diagnosis employing AO (Kawamoto, 1991 b).
A combination of centrifugation, AO staining and fluorescence microscopy was developed into the quantitative buffy coat (QBC) method. Although this technique appears to be easy to handle, sensitive and rapid, it requires costly pre-prepared tubes, a centrifuge and an ultra-violet microscope (Lema et al., 1999). It is not being discussed here in more detail."


Tick-Borne Relapsing Fever Imported from West Africa: Diagnosis by Quantitative Buffy Coat Analysis and In Vitro Culture of Borrelia crocidurae. (1999)
Full text:
  http://jcm.asm.org/content/37/6/2027.full     PDF
"TBRF was rapidly diagnosed for two patients returning from western Africa with fever of unknown origin by quantitative buffy coat (QBC) analysis. ... By QBC analysis of blood from both patients, brightly luminescent spirochetes were observed (Fig.1), concentrating at the plasma-leukocyte interface. Thick blood smears were stained with Giemsa’s stain, and 200 oil immersion fields (×1,000) were systematically examined. In thick smears from the second patient, spirochetes were visible, whereas thick smears from the first patient were negative, even after extensive reexamination."
In the QBC method (see drawing above), by using a capillary tube which comes pre-coated inside with acridine orange and anticoagulant, and where a plastic float forces all the surrounding cells into the 40 micron space between its outside circumference and the inside of the tube, any orange fluorescence in the top of the erythrocyte layer under the buffy-coat layer, makes it faster and easier to detect, if there are any parasitized RBCs, like malaria or piroplasma (babesia) in the sample ...
QUOTE: "Studies have shown that the QBC Malaria Test is 5.5 to 7 times more sensitive than Giemsa thick films. The QBC Test also offers equivalent specificity with a negative predictive value of greater than 98%. The sensitivity of the QBC Malaria Test is particularly notable in cases of low parasitemia, as the test allows for the detection of as little as 1 parasite per µL of blood.  In one study of 49 patients with low parasitemia (defined as <10 parasites per µL of blood), the QBC Test established diagnosis earlier than thick film in 47% of cases."

How does Acridine Orange stain enhance detection of spirochetes and parasites? - some illustrative pictures show it:  

Borrelia (and probably other spirochetes?)
stain yellow to red-orange (single stranded DNA and RNA) with Acridine Orange - examples:


Borrelia hermsii
CDC B hermsii
http://wwwnc.cdc.gov/eid/images/01-0198-F2.jpg

Borrelia duttonii
Borrelia duttonii acridine orange
http://upload.wikimedia.org/wikipedia/de/b/be/Borrelia_duttoni.jpg
copy saved locally: B-duttonii.jpg 

Borrelia burgdorferi
MacDonald-Bb-Acridine-Orange
stained with Acridine Orange, by Alan MacDonald from
http://www.molecularalzheimer.org


Borrelia burgdorferi - cystic form
Bb-cyst-ao-brorson-tigecycline
from Brorson et al. Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline
http://www.pnas.org/content/106/44/18656.full.pdf

Brorson-Borrfelia-cyst-Acridine-orange
from http://www.molecularalzheimer.org

Trypanosoma

http://www.finddiagnostics.org/export/sites/default/programs/hat/images/Trypanosomes_40x_Acridine_orange.jpg
http://www.finddiagnostics.org/export/sites/default/programs/hat-ond/images/Trypanosomes_40x_Acridine_orange.jpg
http://www.finddiagnostics.org/programs/hat-ond/hat/parasite_detection/fluorescence_microscopy/

Malaria

QBC-P_falciparum.jpg
http://www.qbcdiagnostics.com/products/fm/malaria/files/malaria_appnote_pla.pdf

Another fluorochrome stain
Tuberculosis in Auramine O stain, not Acridine Orange!
http://www.zeiss.com/C12567BE00472A5C/GraphikTitelIntern/Tuberkel_fluo_big/$File/Tuberkel_fluo_big.jpg
http://www.zeiss.com/C12567BE00472A5C/GraphikTitelIntern/Tuberkel_fluo_big/$File/Tuberkel_fluo_big.jpg

Leucocyte total and differential count
 should be done on an unconcentrated sample i.e. thin blood smear from capillary finger or ear prick or anticoagulated venous blood, since that gives additional information of any ongoing blood disease. The RBC overview picture may show many cells of abnormal shape (like acanthocytosis, ecchinocytosis), polychromasia (immature RBC which has lost nucleus stains darker bluish, due to content of RNA, compared to mature RBC), hypochromia (less red stain, sign of anemia), increased number of red cells with Howell Jolly bodies inside etc. ... you need to know what all this looks like, so you better have a hematology picture atlas ready at hand to compare what you see with, when beginning to do blood microscopy!  

When looking at peripheral blood cells - http://lymerick.net/Blood-Cells.pdf - the microscopist must be well aware of all the different blood cell types and of the dynamics of the blood cell formation in the bone marrow, i.e. that in response to any increased use, i.e. loss of blood cells, the bone marrow will normally respond  by producing many more new blood cells and will send more new immature cells (blasts .. myelocytes .. immature WBC), normoblasts (immature RBC, still containing a nucleus) into the peripheral circulation, compared to what is seen under normal i.e. non-disease, condition
- hence the presence in peripheral blood of increased number of immunature blood cells, indicate increased production as sign of increased use / loss of blood cells .. the cause of this must be searched thoroughly for - of course!
Normoblasts is a very rare finding in normal adult blood, so if one spot just one of these, think blood loss and lookout for possible cause: bleeding, hemolysis, parasites?
In grave infection the WBCs will usually show signs of activation,
enzyme production i phagocytes result in vacuolization and in toxic granulation of the granulocytes etc.  In very severe infection the number of immature white blood in peripheral blood may be so high that it may nearly mimic a leukaemia blast crisis, it is called leukemoid reaction. When you look at blood smears you might occasionally encounter a leukaemia patient and should be able to differentiate! 
The level of parasitaemia (% is the number of infected blood cells out of 100 blood cells counted in a peripheral thin blood smear) can be so low, that the patient does not develop anaemia, nor are there any overt sign of hemolysis in the urine when the haptoglobin binding capacity for hemoglobin is not yet reached, luckily no risk of kidney damage at so low microbial level. It is the immunreaction raised against the infection that cause disease symptoms, more than the parasites themselves, and since the first line of defence is complement activation and cytokine storm, that are cascade reactions, just a few bugs can sometimes elicit a huge innate immune response. Compare to an allergic reaction, where minute amount of an allergen in the sensible person, can elicit a deathly anaphylactic shock reaction, or in toxic shock syndrome, where toxins are formed by bacteria growing on the mucous membranes, can be absorbed through the membranes and elicit a deathly cytokine response, despite there is not even an invasive blood infection.   
Hemoglobinuria detectable on urine stix may first appear at about 1% parasitaemia and visible hematuria - blood pis - may occur at about 4+% parasitzed cells (is sign of glomerular damage?) in human Babesia infection, judged from reading pulished literature.  Many low-level Babesia carrier patients have less much than 1% of their red blood cells infected (more like only 1 in a 1000 cells infected), i.e. the parasites can be quite difficult to detect by conventional thin and thick blood smear technique and even PCR; as illustrated herein a higher sensitivity and less time to first detection is achieved by upconcentration the blood sample by centrifugation; the buffy-coat fraction of the sample is then used to make smears from and since there is not normally any nucleus or remains of RNA in the mature red blood cell, staining the DNA/RNA by acridine orange may help to quicker detect the intracellular parasites, trophozoites and ringforms, plus also stain DNA/RNA of many other bugs like Borrelia etc.  ...
In 1000x magnification it is possible - by turning the focus handle up and down - to detect if the suspected foreign object is located intracellular (both the object and the cell membrane stands equally clear at the same focus level), or if the object is located under or over the cell, where either the object or the cell membrane stands clear.
With my experience from 10 years of blood microscopy, I can NOT recommend looking for parastites or microbes at less than 1000x magnification, nor on conventional thin and thick "malaria" smears! - is simply not sensitive enough to detect low level parasitaemia! - this is also supported in the published literature.
For detecting borrelia and ringfoms I always use the 100x oil QBC Paralens objective, with normal light source primarily, and as I have 6x and 10x ocular sets for my microscope, I can screen a sample faster in 600x, and whenever I find something that need a closer look, I shift to 10x ocular and view the same area in 1000x without moving the object, and can  exchange one of the oculars for the microcam that connects to my computer via USA, which allow me to take pictures or videofilms of moving objects, like the spirochetes. If I had allowed the sample to stain with a fluorescent dye like acridin orange or react with microbe specific FITC-labelled antibodies before the microscopy (remember to protect the sample from exposure to light or the fluorescence may fade away, resulting in a false negative) - I can switch fast and easy between the light and the fluorescent microscopy modes, by turning off the normal light and turning on the Paralens external UV light.  

Some links on description of immune system / immune cells:
Immune system (WIKI): http://en.wikipedia.org/wiki/Immune_system
Neutrophil granulocyte: http://en.wikipedia.org/wiki/Neutrophil_granulocyte
Toxic granulation: http://en.wikipedia.org/wiki/Toxic_granulation
Monocyte: http://en.wikipedia.org/wiki/Monocyte
Blast crisis: http://en.wikipedia.org/wiki/Blast_crisis
Leukemoid reaction: http://en.wikipedia.org/wiki/Leukemoid_reaction

Microhematocrit technique description (old desciption using capillary tubes):
http://wps.prenhall.com/wps/media/objects/684/700987/ch07HEMA.pdf

QBC method:
http://www.mdinventions.com/successes/envtests/qmalaria.html
"The QBC Malaria method is the simplest and most sensitive method for diagnosing the following diseases.
Malaria
Babesiosis
Trypanosomiasis (Chagas disease, Sleeping Sickness)
Filariasis (Elephantiasis, Loa-Loa)
Relapsing Fever (Borreliosis)
Some research references are shown here. "

Fluorescence microscopy equipment (affordable):

ParaLens (QBC diagnostics) - http://www.qbcdiagnostics.com/products/fm/pla/fab.asp ... 
http://www.qbcdiagnostics.com/products/fm/malaria/files/malaria_appnote_pla.pdf

- is a microscope attachment designed to provide the benefits of flourescence microscopy to any light microscope.
The old Paralens system which I use (PDF printout from wayback machine) have a rather expensive lightbulb of short duration as the light source, and a fiber optic cable connecting the rather heavy external light source to the special QBC objectives, which comes in both 60x oil and 100x oil, while the new Paralens Advance (right picture) system (PDF printout) has a much more durable LED light source, and is much smaller, light weight, more handy, and different power source options. The ParaLens Advance Portability Pack includes options such as a solar battery pack, USB cord, 9-volt battery clamps and more, while the QBC Mobile Power Station is designed to power the ParaLens Advance as well as other QBC products, with a 22 Amp-Hour rechargeable battery and AC/DC inputs. Thus, the system is ideal for ind the field work!
QBC Paralens old   old versus new Paralens QBC Paralens new

LUMIN LED fluorescense - looks like nearly identical QBC Paralens-Advance copy? (who holds patent?):
http://ledfluorescence.com/index.shtml
http://ledfluorescence.com/fluorescencemicroscopy.shtml
http://ledfluorescence.com/diagnostic.shtml 
"Fluorescent antibody (FA) technique, also known as immunofluorescence, is an excellent rapid diagnostic method. FA is easily done, sensitive, specific, and relatively inexpensive. It is extremely versatile. FA detects and identifies both etiologic agents of disease (direct FA ~DFA) and host antibodies (Indirect FA, IFA). A wide variety of infectious diseases can be rapidly diagnosed by FA. Kits and reagents for FA tests are commercially available, and many use highly specific monoclonal antibodies." ...
"
Commercially available monoclonal antibodies can be used for doing indirect FA rapid tests to diagnose other infectious diseases (fluorescent anti-globulins commercially available). Some of these are: (see table on the website)" ...
"Polyclonal antibodies to these etiologic agents and others are also commercially available. Many of these are suitable for both direct and indirect FA tests."

FITC-labelled Borrelia burgdorferi antibodies
 can be purchased commercially from KPL:
http://www.kpl.com/catalog/productdetail.cfm?Catalog_ID=1&Category_ID=144&Product_ID=702
Description: Affinity purified polyclonal antibody to Borrelia burgdorferi made in Goat and labeled with fluorescein isothiocyanate (FITC). Isolated from a serum pool of goats immunized with heat killed whole cells of Borrelia burgdorferi. The antibody is highly specific for Borrelia burgdorferi. Cross reactivity to Borrelia hermsii, Borrelia coriaceae, and Borrelia anserina has been minimzed through extensive affinity adsorption. Product is in lyophilized form. Each lot is tested to assure specificity and lot-to-lot consistency using KPL's in-house ELISA assay.

Zeiss - Primo Star iLED fluorescence microscope:
http://www.zeiss.de/C1256D18002CC306/0/969C99940105AB12C1257489004079FC/$file/60-2-0017_e.pdf

Euroimmun - EURO-Star III plus fluorescence microscope:
http://www.euroimmun.de/index.php?id=produkte_geraete_software&L=1 
http://www.euroimmun.de/fileadmin/template/images/pdf/YG_0301_I_UK_A10.pdf
"A camera can be fitted to the phototube for digital image recording. Switching between the camera and the eyepieces is unnecessary due to the convenient 50/50 beam splitter. For the display and management of digitally recorded fluorescence images we offer the efficient EURO-Picture programme. The standard EUROStar Ill Plus is equipped with a halogen lamp for normal transmitted-light microscopy in bright-field and dark-field and can be upgraded for phase contrast."

SO - in conclusion with modern equipment using a LED light source of low energy demand and very long durability, it is no longer extremely expensive to get a microscope system that can do fluorescent microscopy plus light / phase contrast / darkfield.


The challenge is to find a to the microscope suitable and wellfunctoning microscope CAMERA that can connect easily to and be powered by any computer via USB!
It must have a fairly high resolution and not the least must be  LIGHT SENSITIVE ENOUGH to make it possible to take nice pictures and/or videos as illustrated. During microscopy the video can run and copy all seen, snapshots and videoclips from the raw film can be extracted later.  This makes it much easier to find microbes, even a very thin/slim spirochete, that is either stained orange-red by Acridine Orange (cheap, see pictures above) or green with FITC-labelled Borrelia specific antibodies (see below) .. - photographing a single green spirochete up against a very black background is very camera demanding, I can unfortunately NOT photograph a slim green spirochetes on a black background with my camera, despite it is just possible to see such in the microscope!

Affordable microscope cameras like the DinoLite series comes in many different versions; I now use the DinoLite AM4023X for my old Leitz Laborlux III microscope anno 1957, because I do not have the trinocular top for it; but - despite it has a higher resolution of 3 Mpixel - it is unfortunately not quite as light sensible as my old Bresser Microocular II camera (pic), the very big problem with my old Bresser microcamera is that is has only VGA resolution (640x480 pixels) and worse, there are only 32-bit drivers for this 32-bit device, i.e. no drivers can make it work on a 64-bit Windows (Vista-64 or 7) system, therefore I had to buy a new camera after shifting to a Windows7 computer system!

Plus of course one need good software i.e. a picture and video grapper (can be found as free download) and editor program (free Windows Movie maker is okay, but lack marking tools and can only save video in WMV format) - one need at program that make it possible to measure and mark structures, can put on arrows, rings, and text remarks on both still pictures and video etc.  The DinoLite software works very well, but the program only works with DinoLite cameras of any type.
Note also the hand or stand held DinoLite microscopes which magnification ranges from ~10 to 200 to 500x, depending on the version; LED lights are built in in white, polarized, ultraviolet, infrared or a switchable combination. Most versions have calibration and measuring options. Its housing is either composite or aluminium alloy ...
Very nice "toys" that can be used for many purposes at affordable price, like photographing ticks  ... what will future bring of enhancements?


Borrelia:

Borrelia burgdorferi specific direct fluorescent (FITC) immune stain
:    

BB31 direct immune stain Alan MacDonald
Picture taken by Alan MacDonald (ca. 1985), borrowed with permission.
Both spirochete and cyst form and "granules" stain with Borrelia antibodies!


Embers-DFA Borrelia burgdorferi
From Embers et al. (Full text):
Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection (2012)
Figure 5. Spirochetes recovered by xenodiagnosis from animals treated in the disseminated phase of infection.
Images from direct fluorescent staining of B. burgdorferi spirochetes found in xenodiagnostic tick midgut culture (A)
or tick midgut preparation (B) from treated animals GA59 and GB56, respectively.


Note that the above Borrelia burgdorferi spirochetes do not look very spiralled
(In TBRF pictures the spirochetes often seem more coiled than B. burgdorferi).

BUT all those who claim that Borrelia burgdorferi spirochetes has so and so many coils
and is always of a certain length and thickness
- and say that what I call spirochetes do NOT look like ideal Borrelia spirochetes from pictures
- have obviously not spend years studying the morphology and
movements of live spirochetes in the microscope themselves!

Enjoy especially the amazing movies at (Youtube searching for Lymebugs videos)
- all microscopies were made by Stan Dembowsky in 1999 from his studies
of the
laboratory grown spirochete Borrelia burgdorferi B31
... demonstrate:
Borrelia uncoiling itself and changing morphology at its will pretty fast
Growth by division, growth in colonies within biofilm like substance,
adults spirochete form and baby-spirochetes, blebbing, cyst form ..
Growth of Borrelia B31 on sheep blood agar, where baby-spirochetes
seem to be emerging from the surface of the red blood cells?
Stan also studied the effect of penicillin on Borrelia B31 
...

CDC on Tickborne relapsing fever borreliosis (TBRF):  http://www.cdc.gov/relapsing-fever/clinicians

The very important Borrelia relapse pattern:
TB-RF timeline (CDC)
Note that spirochetes are only visible in the blood during the symptomatic episodes, but the blood was still (more) contageous also in between the relapses, when there is no visible spirochetes in the blood:
http://lymerick.net/1914-Nicolle.htm
=> CORRECT TIMING of sampling of blood specimen for microscopķc examination for spirochetes in relation to the patient relapse cycle (during attacks) is essential, if one  does not time the sample right there is a high risk of not seeing any spirochaetes!
In case the patient has a cycle that runs over 10 days, we only have 10% chance of doing it on day one in a new relapse, if one just take a random sample, and NOT time the sampling according to the relapse cycle; in case a patient has a monthly relapse pattern the chance of finding spirochetes is 1/30 i.e. 3%! 
I only have greater "luck" finding spirochetes by microscopy, than most other investigators, because I understand the relapsing pattern, and plan after it; I ask the patients to do a very detailed Excel symptom diary (OA but respect my ownership, dont remove my name) and from the relapse cycle I can deduct, when is the best time to see the patient and do sampling for direct test for spirochetes! - and can judge effect of any intervention.

A immunocompetent relapsing fever Borrelia patient will typically get from 10-30 relapses, before the disease "burn out" when the immune system achieves control over the microbial proliferation  .. probably likewise for most patients infected with Borrelia burgdorferi?
Symptom flares (attacks) are gradually diminishing in severity (relating to the amount of spirochetes that enters the blood and raise complement cascade reaction (you should measure complement split products if available) and proinflammatory cytokine storm (TNF => fever, IL-1, IL-6 ...) and late in the course, also the intervals between relapses is increasing, as long as the infection is kept under control (by immune system and/or antibiotics) then newly formed spirochetes are not allowed to reproduce themselves weekly any more ...

It is usually not mentioned in the literature that Lyme borreliosis also clinically gives a relapsing infection pattern, just like the close relatives in the relapsing fever Borreliosis group (except TBRF is perhaps more pathogenic, cause a higher TNF (fever) response than the Lyme borreliae does in the immunocompetent host?).
BUT there are some works examining this subject  ..
In very ACTIVE LATE (> 6 months post infection) Lyme borreliosis, where moving spirochetes can repeatedly be found in buffy-coat blood fraction from the patient by microscopy, but usually only during the first day of a new flare, the clinical relapse pattern is the same, weekly relapse pattern, as descibed in TBRF!
Whenever the borrelia infection is under relative control by the immune system and/or antibiotic treatment, the relapse cycle shift pattern, usually within 4-6 weeks after start on antibiotic treatment, from the previous weekly to a monthly cycle at 3-4 weeks intervals!
The monthly clinical relapse cycle was  described by dr. Joe Burrascano in his diagnostic guidelines, already since the early 1990ies, see ILADS: http://ilads.org/lyme_disease/treatment_guidelines.html

Cyclicity was also noted by Willy Burgdorfer  ...
Lyme borreliosis: a relapsing fever-like disease? (Burgdorfer 1991) 
http://www.ncbi.nlm.nih.gov/pubmed/1947807
... [for up to 3 months after animal aquired the Borrelia infection feeding] ticks were evaluated for spirochetal infections by direct immunofluorescence. All mice were found to circulate spirochetes for at least three months in concentrations sufficient to infect ticks. The percentage of infected ticks alternated from low to high, suggesting occurrence of episodes of mild and heavy spirochetemias. The results suggest that B. burgdorferi in its animal hosts and possibly also in humans causes prolonged spirochetemias characterized by episodes of alternating high and low concentrations of spirochetes as reflected by similar percentages of infected ticks. The long persistence of spirochetes in the peripheral blood stream and the cyclical form of Lyme borreliosis appear to be related, as in relapsing fevers, to the capacity of B. burgdorferi to undergo antigenic variations."

The two relapse patterns, that are both characteristic of clinical Lyme borreliosis, are probably well explained by Brorsons laboratory culture studies, finding that it take the YOUNG cystic forms of Borrelia about 9 days versus for OLD cystic forms about 4 weeks to reproduce the spirochaete form:
http://lymerick.net/1998-Brorson.htm


The patients with VERY ACTIVE, weekly, RELAPSE pattern usually will respond to antibiotic treatment in a quite typical and gradual way, as illustrated by symptom log curves from case#24, who kept my Excel symptom diary (early version); there are 7 days in between the dates on the X-axis; arrows indicate the circa weekly clinical worsenings (6-14 days acc. to old research on RF-spirochete lifecycle done a century ago):
Borrelia burgdorferi - antibiotic treatment response
The monthly relapse cycle (some more intensive than others) is illustrated by curve from project patient #11 (still one week in between dates on X-axis):
Borrelia burgdorferi - monthly relapse cycle
More on videomicroscopy for spirochetes and the Excel symptomdiary (free download):
http://lymerick.net/MK-videomicroscopy.html 
My studies done before 2006 using direct fluorescent immunostain with borrelia specific antibodies from KPL (done as research in BRT), and primitive cheap equipment: http://lymerick.net/videomicroscopy.htm  
Other reports on chronic (late diagnosed) or persistent (post antibiotic treatment relapse) borreliosis documented by positive culture, microscopy and/or PCR: http://lymerick.net/persistent-borreliosis.htm

Borrelia culturehttp://lymerick.net/Borrelia-culture.html  
Culture in commercially available BSK-H medium* is often successfull, especially when there are a few visible spirochetes in the wet drop sample from a fresh buffy-coat prep.; after positive culture much more material can be examined by other methods, i.e. there is a possibility of PCR typing of the Borrelia strain when there is enough spirochetes. DON'T THROW MATERIAL OUT AFTER ONE MONTH as some researchers do, according to published reports on culture. In adverse conditions it may take much longer  for slow Borrelia to grow in vitro: 
http://lymerick.net/Borrelia-growth-optimum.html

*) Sigma-Aldrich http://www.sigmaaldrich.com/catalog/product/sigma/b3528?lang=en&region=DK

I read (but forgot in which ref.?) that some found that Borrelia cyst may not grow spirochetes well, unless they have experienced a colder period, like they would have experienced in a natural life situation, when residing in dormancy in a tick during the winter-time. Can the microbes sense the seasons somehow?
Many chronic patients (that are not in antibiotic treatment), descibe getting longer worse periods, shifting to a weekly relapse pattern during spring-time (late march-mid june) and during autumn (mid-august to late november), while they shift to a monthly relapse cycle during high summer (high temperature, low moisture so ticks are not questing), and monthly cycle during winter time ...
- can tickborne microbes sense somehow the seasons when ticks are active questing for blood meal out in the nature, and become active then, while they go into dormancy at times when the chance of a tick biting it host is lower?  


Please note, that all microbes that use the TICK - with ½-1 year between blood meals - as vector, ALL have the ability to go dormant in the tick for at least six to twelve months, otherwise they would not be able to keep the infectious tick-mammal-tick life cycle going. The microbes also go dormant inside their mammal host, when the environment becomes unsuitable for the spirochete form, like when the patient forms lots of antibodies, under antibiotic treatment, lack of nutrition, wrong pH, too high oxygen etc; persisters can survive common treatment and some cnb awaken later when the environment again favours regrowth of spirochetes, that has been dormant for a very long period.
This was observed by Hampp already before 1950! - http://lymerick.net/1948-Hampp.htm   
"Typical free granules, the end products of granule 'shedding', are shown in figure 18.
They are roughly circular in outline and sharply bounded. They consist for the most part of what appear to be short sections of spirochetes closely packed together. The contents of these granules are probably responsible for the fine lacelike appearance and the bright white, highly refractile bodies described by Hampp (1946) under the dark-field microscope.
Examples of another type of free granule repeatedly observed are shown in figures 19 and 20. These granules consist of tangled masses of spirochetes or spirochetal segments.
The significance of granules in the life history of the spirochetes is unknown but certain investigators have suggested that they may be germinative units (Balfour, 1911; Noguchi, 1911; Noguchi, 1917; Leishman, 1918; Mudd et al., 1943; Hampp, 1946). Others are undecided or hesitant in accepting this hypothesis (Fantham, 1916; Akatsu, 1917; Wenyon, 1926; Warthin and Olsen, 1930). Topley and Wilson (1936) have indicated that they are probably particles of culture medium adhering to the sides of the spirochetes. The electron micrographs demonstrate that this explanation is wrong, and that free granules are definitely a phase in the development of spirochetes. Although it is not possible to determine from these micrographs that the granules are germinative units their constant rhythmic occurrence in living cultures suggests this possibility. Further support of this hypothesis is provided by the fact that cultures up to 31 months old, showing only refractile granules by dark-field examination have invariably given normal growths on transfer to fresh medium (Hampp, 1946)"

31 months is circa 2 ½ years!  

DualDur reagent, Dr. Bozsik patent description (2004) - double centrifugation technique (removes blood cells):
http://www.patentgenius.com/patent/6689577.html
"In our experience, it is still possible to detect the pathogenic bacteria if there are less than 10 bacteria in a milliliter of centrifuged native blood samples. In comparison, the threshold for the detection of Lyme borreliosis with PCR, which is currently considered the most sensitive but can only be done in specially equipped laboratories, is between 40 and 100 germs per ml; besides, as many as possible primers specific to different sub-strains should be available."

[20 drops per ml, i.e. 1 drop is 50 microliter, i.e. there may be one spirochete per 2 drops of HIGHLY CONCENTRATED BLOOD sample, after double centrifugation! .. of course one will need to examine more drops in order to find the spirochete, if there are so few - the investigator must be very meticulous!]

Detection of Borrelia in acridine orange-stained blood smears by fluorescence microscopy. (1983)
http://www.ncbi.nlm.nih.gov/pubmed/6602602
"We concluded that the AO stain is simple, rapid and more sensitive than Romanowsky methods for detecting cases of low-level spirochetemia."

SHORT REPORT: Detection of Borrelia (Relapsing fever) in rural Ethiopia by means of the quantitative Buffy Coat Technique. (2001)
http://www.ajtmh.org/content/65/2/164.full.pdf
"In laboratory studies that used Borrelia burgdorferi as a model, we detected spirochetes at concentrations as low as 10 organisms/mm3, whereas the number of positive readings assessed by means of stained blood films fell significantly at dilutions below 3,263 organisms/mm3. The greater sensitivity of the QBC technique is important in areas where Borrelia is endemic. .."

Note 10 / mm3 = 10 /  microliter, i.e. correspond to 500 organisms per examined blood drop; number of spirochetes in RF is much, much higher than in Lyme borreliosis, where > 7 organisms per drop is "many"! 

Babesia:

Phylogeny and evolution of the Piroplasmida as inferred from 18S rRNA sequences. (2012)
http://www.ncbi.nlm.nih.gov/pubmed/22429769
The order Piroplasmida consists of several genera of tick-borne parasites that infect mammals, and to a lesser extent birds, and are therefore of medical and economic importance. Despite their importance, considerable confusion exists concerning the relationship among piroplasmid species, specifically concerning the number of genera and the intergeneric relationships.
To examine evolutionary relationships among piroplasmids, we conducted phylogenetic analyses of 192 18S rDNA sequences from the genera Theileria, Babesia and Cytauxzoon.
Our analyses revealed eight clades potentially representing distinct genera, and we distinguish the Duncani Group and Microti Group as genetically distinct groups of species requiring detailed analysis of morphology and life-history to allow formal generic description.
The piroplasmid phylogeny revealed considerable host diversity and limited host specificity, suggesting piroplasmids have undergone frequent host switches during their evolution. Our analyses provide the first reported evolutionary timescale for piroplasmids independent of the assumption of parasite-host cospeciation, which is invalid for piroplasmids.
Evolutionary rate analyses revealed considerable substitution rate heterogeneity, which we attribute to host switching and diversification. Finally, we call for a comprehensive phylogenetic, morphological and life-history analysis for these medically relevant taxa to resolve relationships and understand host specificity.

The application of acridine orange staining to quantitate low levels of Babesia divergens parasitaemias. (1974)
http://www.ncbi.nlm.nih.gov/pubmed/4138127     
http://wwwnc.cdc.gov/eid/article/17/1/pdfs/10-0737.pdf

"Our cases highlight that, in Europe, babesiosis can occur in healthy persons and manifest as moderate illness. The rarity of other reported cases in nonimmunocompromised patients in Europe may be related to the difficulty of diagnosing babesiosis. A stained thin blood smear is rarely performed in Europe after tick bite in healthy patients. The difficulty of detecting intra-erythrocytic forms of babesia coupled with frequent low levels of parasitemia, may result in accurate diagnoses, although acridine orange and fluorescent microscopy may assist in the detection of parasites (1). Other diagnostic tests, such as PCR and serologic analysis, are not routinely performed in France and require a reference laboratory (8). ....
Babesiosis, although difficult to diagnose, needs to be diagnosed for various reasons:
1) without treatment, babesiosis can lead to severe illness;
2) the disease can persist for a long period without symptoms, which could lead to posttransfusion cases (12); and
3) effective specific treatments are available (atovaquone plus azithromycin, or for severe cases, clindamycin and quinine) (2).
These drugs are not usually prescribed in febrile tick-bite cases; doxycycline is the usual drug used to treat tick-borne bacterial diseases. Moreover, patients with moderate infection could benefit from an atovaquone plus azithromycin regimen, which is better tolerated (13). ...

In Europe, babesiosis is probably underdiagnosed; thus, we suggest that when patients have influenza-like or malaria-like syndromes after confirmed or suspected tick bites, a blood smear be performed regardless of whether the patient is immunocompromised.
Blood smear can identify not only Babesia spp. infection but also Anaplasma spp. infection, another emerging and underdiagnosed tick-borne illness. In cases of new European Babesia spp. infections, a deeper characterization of the strains by erythrocytes cultures and standardized PCR, as well as a systematic study of the patients’ immune systems, should be undertaken to enable a better understanding of this disease.
"

Babesiosis: recent insights into an ancient disease. (2008) 
http://www.ncbi.nlm.nih.gov/pubmed/18440005

"Most significantly, molecular analysis of the implicated pathogens suggests that the host-range of many babesia is less restricted than believed previously hitherto unrecognised species can cause infections in a variety of animal hosts and in humans (Zahler et al., 2000: Cho et al., 2002: Herwaldt et al., 2003, 2004; Conrad et al., 2006: Kjemtrup et al., 2006: Häselbarth et al., 2007: Kim et al., 2007). Therefore, many past cases of human habesiosis on both sides ot the Atlantic that were attributed, hased on traditional methods, to classic species such as B. divergens or B. microti, may indeed be due to species not yet known to cause such infections in humans (Herwaldt et al., 2003: Gray, 2006: Hildebrandt et al., in press).
This notion is further substantiated by the recent recognition of Babesia duncani [WA1] and B. divergens-like organisms as pathogens of medical significance for humans in the US (Herwaldt et al., 1996; Beattie et al., 2002: Conrad et al., 2006). Moreoven, confirmed autochthonous (~local strains of) B. microti infections have been reported in Taiwan, Japan and Europe (Shih et al., 1997: Saito-Ito et al., 2000: Hildebrandt et al., 2007), and a new European B. divergens-like organism (EU 1), provisionally named Babesia venatorum, has been discovered, which is probably a parasite of deer (Telford and Goethert, 2004: Bonnet et al. 2007). This parasite was involved in the first documented cases of human babesiosis in Italy, Austria and Germany ( Herwaldt et al., 2003; Häselharth et al., 2007). ..."


SUMMARY:
ALL THE OLD ASSUMPTIONS HAVE PROBABLY BEEN WRONG
- since during the last 2 decennials many new types of microbes have been detected after PCR typing began - and genera shows greater diversity and little host specificity within the Babesias, as well as in the other tick transmitted infections!
Humans with ringforms in their red blood cells, appears to have not yet been investigated for if they instead of Babesia, 
could be infected with the closely related other piroplasmida, that infect a variety of animal hosts, Theileria and Cytauxzoon!

Note: It has been proposed by some to move/rename Babesia microti to Theileria microti, based on a closer genetic relationship of this group to Theileria, than to other Babesia spp.  
Theileria infect both lymphocytes and red blood cells, and can form multinucleate intralymphocytic schizonts. More about Theileria in this PDF

    
Below focus is set mainly on two of the newly found Babesia types, first called EU1 and WA1, because they seem esp. relevant to DANISH patients:

1. Babesia duncani (former WA1):
Anti-WA1 antibodies were detected in a high fraction, no less than 14 of 132
(10,6%) danish neuroborreliosis patients, had antibodies reacting with Babesia WA1, an - at the time - very newly in the USA found Babesia variant, acc. to conf. abstract presented in USA 1996. Unfortunately no further danish studies of Babesia were done, acc. to the lead author Anne-Mette Lebech,  they put the work away, thinking it was "all false positives"! ...
There is no smoke without a fire! - if we do not have Babesia duncani in Europe, we do have other Babesia gibsoni-like spp. here, that might infect people and may give rise to antibodies that crossreact with the Babesia gibsoni-like B. duncani antigen?! .. it would be prudent to search for which infection could have raised cross-reacting antobodies!
This should immediately have been properly investigated of course, in ticks, in reservoir animals and in sick humans that did not recover after conventional antibiotic treatment for Borrelia - but sick people get ignored!
MANY, including myself, have been told "we dont have Babesia here in Denmark, so there is no reason to test you for it" - but when we got tested, many had ringforms inside red blood cells, besides borrelia!
In 1998 I asked for test for co-infections, including Babesia, Ehrlichia, but I was denied testing, reason being told "not available in Denmark, and too expensive on the departments budget to send tests abroad"!
I was very sick and soon after had to go on permanent sick leave / early retirement; not getting proper diagnostic help from my danish colleagues, I searched the world for other test options (paid out of own pocket) and was in 2001 diagnosed in USA with certain persistent borreliosis by direct immune stain for Borrelia burgdorferi, very likely babesiosis, since there were ringforms in several of my RBCs, and possible MONOCYTIC Ehrlichia; then I ordered serologies, but Statens Serum Institut (SSI) decided not to send my samples abroad for testing with serology nor PCR for the co-infections, despite there were published reports showing it could have been done; the local microbiology department did (very surprising to me) not ask for serial bloodsmears, so I had to do it all myself. Got the serial smears stained by the local pathology lab. who mounted coverglass on the smear - so later these many serial thin bloodsmears were reexamined by Italian vet. Walter Tarello. The comparison result is reported in my York 2004 presentation. Unfortunately I did not get the smears back :(     
SSI could and should have researched PubMed via Internet. They could have found and read about the method used by the Japanese for detecting babesia i farm animals, that was used in 1999 in the first human Japanese donor transmitted case investigation  - http://lymerick.net/York2004/Japanese-donor-Babesia.htm - which serves us now, as example of very good clinical and research practice!
 
The Japanese investigators had traditionally investigated bovine babesiosis cases by inoculating blood from sick babesia suspected cows into special breed of immune deficient laboratory mice, whose blood had been exchanged with blood from healthy cows and likewise for other animals; the blood exchange procedure creates a much more Babesia sensitive host animal, at test system that can detect infection in very low-level carrier state, below detection with microscopy and PCR, like in healthy blood donors;
they copied the babesia detection method from animals, except for using human blood instead, i.e. the NOD/shi-scid mice circulating erythrocytes (RBCs) had been replaced with human RBCs (hu-RBC-SCID mice) to facilitate "culture" of the parasites in the mice. 
NOTE: The donor's serum exhibited a high antibody titer against the local Babesia microti-like isolate from the patient, whereas it exhibited only a weak cross-reaction against (three) B. microti strains isolated in the United States.    
"Examination by microscopy and PCR failed to detect the parasite in the donor's blood obtained 8 months after the donation of the blood that was transfused. However, we were able to isolate Babesia parasites by inoculating the blood sample into SCID mice whose circulating red blood cells (RBCs) had been replaced with human RBCs."
LESSONs learned:
1. asymptomatic donors can have so low level carrier status of Babesia, that it can not be detected by microscopy, nor by PCR, but still there are enough parasites to pose a risk of transferring infection to susceptible blood recipients via transfusion! - the least one can do is to ask donor about risk factors for aquiring Babesia infection, which is not done routinely by the blood banks!
2. the Japanese (Kobe) Babesia microti-like strain could be detected by US B. microti PCR assay, while the antibody response measured very weak with 3 american B. microti strains as antigens; both patient and donor had high antibody titers against the local variant => the same situation might apply in Europe vs USA, i.e. when danish SSI lab. send samples to CDC - that are using US Babesia strains as test antigens? - these test may not detect the European patients infected with local European variants, who may test very low or false seronegative, due to strain antigen variation.
The best must be to try to isolate and culture local strains and then use these strains as antigens in indirect IFA assay, exactly what the smart Japanese investigators did! 
If the highly susceptible hu-RBC-SCID mice, after inoculation of a suspects blood, develop a very high level of parasitaemia, the blood of the mice can be used in homologe IFA serology test, with the patients and the donors serum.
Local IFA i.e. with locally found antigens / strains can be developed; once the local parasites are catched from patients or local reservoir animals, it should be reasonably easy to maintain the local strains in animal culture in abundance enough to do many IFA tests by copying the Japanese method, using hu-RBC-SCID mice; how to develop a local IFA is explained in detail in the Japanese article in english! 

In USA Babesia duncani (WA1) infection seem to be a rare*, but more serious - also in previously immunocompetent individuals that have not been splenectomized - compared to Babesia microti that is often described as mild (by doctors, patients tell different) and rarely fatal (mortality rate can only be calculated for the diagnosed cases, there may be many undiagnosed cases).
*) VERY IMPORTANT TO KNOW is that Babesia duncani (WA1) parasite 
is genetically distant from and is not crossreacting with B. microti, so Babesia duncani infection is not found by PCR and antibody test for B. microti which is normally the only Babesia test applied in USA! - see references below!
*) rarely diagnosed can be because the right test is not done at the right time in the patients disease course, or because of testing for too different strains from the on infecting the patient!


Strain variation make commercial serology tests that are based on a single strain unreliable, i.e. high risk of locally infected patient only showing borderline positive or false negative antibody result, if a commercial antibody kit is used with foreign strains from distant areas, that are too different in antigenic expression from the local variants.

2. While occurrence of Babesia infection in European ticks is generally found low, with current PCR tests (~1% of ticks), the occurrence of Babesia venatorum (EU1) seems to be relatively far more common in the european tick populations than is B. divergens and other Babesia spp., according to very recently (2010-2012 published) investigations from several European countries, references listed below ..


3. passerine bird routes up to Norway may fly over Denmark and some of them was found to harbour Babesia venatorum (EU1), but not other babesia spp.
The birds may stop for fouraging and resting many times
underways, dropping of or aquiring infected ticks locally, i.e. danish humans and animals are likely at some (low) risk of getting infected with Babesia EU1!
=> therefore danish patients suspect of Babesia infection (ranging from having episodic (4-5 days interval it seem) mild hemoglobinuria (stix can be done by patient at home, and do not cost much) and symptom flares, to overt babesiosis i.e. severe hemolysis resulting in blood-pis, and development of anaemia ... should first of all be investigated for this most likely Babesia type to become infected with ... 

Unfortunately none of the previously reported tick studies from Denmark, examined danish ticks for presence of Babesia spp., so
we DON'T KNOW THE FACTS about prevalence of Babesia tick infection rate investigated in local Danish ticks
- therefore I needed to estimate the risk of getting infected with different Babesia species from other studies done in our neighbour countries in Europa, where the passerine birds may have come from, that later reach Norway via route over Denmark.

Low level carrier infections pose a threat because Babesia and other microbes can be transmitted to very susceptible patients needing blood transfusion! - but do not get diagnosed due to lack of tests, to lack of knowledge / awareness in doctors and the public, or worse by ignorant and arrogant doctors that should have studied the literature to know how the best standard (Japanese case study as teacher), but who chose doing the wrong tests, with too much differing antigens, too low sensibility, test with an - a priori - very low chance of showing any positive results!
Donors are not screened for possible Babesia or other tickborne infections, the blood banks just ask if the donor feel healthy, then okay to tapping; they do not even ask if the donor have had tickbite, known borrelia infection etc. (asymptomatic long term carrier state of babesia is very well documented in published literature) ... I NEED TO DO MY PART TO CHANGE THAT, BY INFORMING AND RAISING AWARENESS!

This little "review" was prompted by a very recent investigation of a danish chronically ill borreliosis patient (rheumatologic "polymyalgia rheumatica" and started corticosteroid treatment, that were later diagnosed with repeated episodes of hemoglobninuria on stix
- the patient had a weak / borderline seropositive (1:128) in a Babesia serology test, where the test antigen used was not revealed in the report, but it was probably a European B. divergens assay (since that is what is usually tested for in Europe)?
- i.e. possibly it could perhaps be a weak sero-cross-reaction with the Babesia divergens-like Babesia venatorum (EU1) if the patient has this variant? .... it would probably NOT give any cross reaction, if the patient was infected with the Babesia gibsoni-like B. duncani / WA1, who is more distant from B. divergens  ..

Babesia venatorum (EU1) is probably the most likely Babesia species for humans and animals to become infected with here in Europe, judged from a higher occurrence of this strain in the European ticks, than B. divergens and B. microti, reports coming from several areas of Europe - all areas where Babesia has been investigated so far - this is illustrated in selected articles below on Babesia venatorum (EU1) strain!
Weeks later the patients serum was send to CDC, who applied an B. microti assay (probably using a US B. microti antigen variant, which can be problematic acc. to Japanese donor transmitted case, if the patient has a local Babesia microti-like variant, plus an experimental B. divergens assay .. relevant question is, did CDC use USA variant of B. divergens, found in Washington, which may differ from EU B. divergens?) OR did CDC use a European B. divergens variant, that could possibly show borderline positive cross reaction, if the patient is truly infected with B. venatorum? ... both CDC assays came out negative more times ..

Thereafter the danish microbiology reference laboratory, Statens Serum Institut (SSI), recalled the previous seropositive as probably having been a false positive serology, which they can NOT, really!
I detected nice ringforms in this patient blood (ON43) blood on Nov. 9, 2011, by microscopy of buffy-coat smears plus I also found moving spirochete-like structures in wet drop prep. from buffy-coat fraction, see video (more pictures of ringforms therein too.
The 
written report in danish (with pictures and link to video) was immediately send to the ID doctors in charge of the patients care at the time, so I did my part to help the patient get diagnosed properly (I am just a consultant for a danish patient org. with special interest in direct diagnosis of tickborne infections, doing a little bit of research on my own)!
Written report in danish: http://case.ulmarweb.dk/ON43/ON43-20111109.pdf    Video:  http://case.ulmarweb.dk/ON43/ON43-20111109.wmv
BUT THE MICROSCOPY FINDING WAS APPARENTLY IGNORED! - it is not even mentioned in the University hospital infectious disease doctors papers on the case, they don't count in my microscopy finding in their conclusion on this patients case, whom they later dismissed as "never had babesia", based on neg. serology and PCRs that could have been false negative, if not using the right strains as test antigens / using wrong primers for PCR!
-- the patient was offered no treatment neither for ringforms nor spirochetes, except CORTICOSTEROIDS, a perhaps dangerous treatment for the patient! 
None of them answer the patients and my good question: which other Babesia-like antigen could then have produced a false positive serology in the patients?  

Furthermore, the very same day I did extremely thorough blood microscopies on buffy-coat fraction from the patient and found ringforms in more of his buffy-coat smears, the ID department in the University hospital send this patients PLASMA for Babesia PCR (unknown primer), and with negative outcome, OF COUSE! ... since a positive outcome of PCR could not be expected, because Babesia parasites resides inside the red blood cells, which are NOT PRESENT in the PLASMA FRACTION they send for PCR !!!!

The doctors responsible for care of this patient chose to conclude that this patient never had babesia infection! ... which they can NOT, really, based on the investigation they have done, because they have not done  specific test for Babesia duncani (WA1) nor for Babesia venatorum (EU1)! ...

False negative PCR test results can be a result of very low degree of parasitaemia
(like in the Japanse donor 8 months post infection of a blood recipient) and perhaps the degree of parasitaemia may fluctuate over time, it is probably way under 1%, since it can not be detected on conventional thin and thick smears, nor by PCR AND/OR due to use of the wrong test, i.e. they only tested for Babesia microti and Babesia divergens (both USA variants?), and used negative results to dismiss the previous borderline positive Babesia titer.
My guess is, that this patient could have a Babesia venatorum EU1 infection cross-reacting minimally with B. divergens as test antigen?
 
NEITHER SSI (nor CDC) HAVE BOTHERED TO INFORM US IN THEIR TEST REPORTS EXACTLY WHICH BABESIA ANTIGENS AND PCR PRIMER THEY USED IN THE NEGATIVE TEST!
NECESSARY INFORMATION IN ORDER TO REMOVE ANY "REASONABLE DOUBT" THAT THE RESULTS COULD ALL BE FALSE NEGATIVE, BECAUSE THEY CHOSE WRONG STRAINS WITH LOW RESEMBLANCE TO LOCAL STRAINS!

This patients (and all other suspected) Babesia case(s) should be examined just as thorough as the Japanese index case was investigated 10 years ago, i.e. by inoculating the patients blood on SCID-hu-RBC mice, and if parasites can be cultured this way, serology must be done via IFA using the patients own strain as the test antigen, just like was done in the Japanese case!    

Many doctors have told danish patients "we dont have it here, so there is no need to test you for it" and have dismissed "borderline" serology results as false positives ...
There is no smoke without fire! .. a borderline means something that need further investigation, not to be dismissed as if it could not be of any causal significance, especially not if the patient is chronically il, without any other reasonable explanation found for the chronic illness - it would be prudent to investigate properly - thinking "out of the box" on uncommon or even new infections, when the patient present like this one, with repeated episodes of hemoglobinuria, and concurrent fever and elevate CRP!

The implication of we now have recognised that passerinee birds may carry ticks over several thousands of kilometers from Africa to Norway, that may be infected with multiple human pathogenic microbe species, that may be transmitted to pets and people along routes of the flights and CAN make the animals very sick - explains why neither BORDERS nor FENCES can contain infections and prevent the spread; if we don't have it here already, it is very likely just a matter of time before we get it ... "WE DON'T HAVE IT HERE" is probably a very wrong statement! ... we do have it, but need to do the right tests to find it!

PROPER CASE INVESTIGATION AND COMMANDED SURVEILLANCE OF OCCURRENCE OF ALL THE TICKBORNE INFECTIONS IS NECESSARY, BETTER LATE THAN NEVER!
- using the very most sensitive diagnostic methods of course!


4. Personally I am quite sure we already have more Babesia spp. here in Denmark already (vets recognize that cows get Babesiosis sometimes), and probably more types of Babesia, just like in our neighbour countries ...

I, personally, was the very first human IN DENMARK AQUIRED CASE, that was diagnosed with ringforms by simple microscopy of thin bloodsmears back in 2001, which prompted me to researh this issue as much as I can!

I send the results to the local microbiology dep. as well as to SSI and asked for proper evaluation, the response from the local microbiology dept. "it is too controversial".
SSI offered inoculation, culture and PCR for BORRELIA only (not mentioning anything about how to diagnose the co-infections) IN CASE I RELAPSED AFTER TREATMENT, which was not yet begun, because the ID professor demanded certain proof for all infections via a danish microbiologist, before he would offer me any treatment for the three infections!
... when I did relapse badly, with spirochetes in my blood found twice within a year 2008 - by two different investigators and 10 months apart - I asked again, both the local microbiology dept. and SSI for the test they had promised me back in 2001; the head of the local microbiology dept. answered in wrtiting: no knowledge, no expertise in our dept., no ressources, NO INTEREST! ... SSI "we do not do the test you ask for any more and have no plan to do these methods in the future"! - so THERE is no help for danish chronically ill patients, getting direct test for Borrelia is NOT POSSIBLE - therefore I HAVE TO DO MY BEST to help sick people!  
       
A danish saying "Need teaches a naked woman to make clothes" ... because I did not get proper diagnostic help myself by my danish colleagues, was denied tests described in the published literature, I was forced to "do-it-myself" and to find another way, with help from good colleagues abroad, and the experience gained from this, led me to do a little research project on patients with similar history and symptoms.

In York UK 2004 Lyme conference I personally reported on 15 danish case
of ringforms detected in thin blood smears from until then 33 enrolled projects participants, who had Borrelia antigen in their blood also, detected by direct immune stain for Borrelia burgdorferi sensu lato: http://lymerick.net/York2004/ringform-DK.pdf ... (I am #1)
Overall about 1/3 of a total of 50 project patients with "chronic Lyme" symptomatology investigated, also had ringforms in their RBC, and 75% of the 33 first "pilot project" participants had microscopic sign of one or more co-infections PLUS borrelia antigen in their blood detected by direct immunofluorescent stain specific for Borrelia burgdorferi sensu lato ... it was just a small pilot study with few patients (but what I could do with very low economic and practical ressources, being sick myself on top), and it was not a treatment study, but as some patients recorded all the treatment and symptoms (and many also came for repeated blood microscopiesby me) during the course of treatment via Excel diary with automatic curve drawing, the improvement on treatment could be calculated in these (too few) cases; those who got relevant antibiotic treatment for BOTH found co-infections AND borrelia IMPROVED OVERALL 50-75%, usually within 3 months, some had further improvement later; patients sick less than two years had a good chance of full recovery, but many had been sick for a very long time, overview over pilot project participants from the UK 2003 lecture: http://lymerick.net/York2003/projpatients.pdf

This prompts for further investigation, which will cost a lot of money we dont have, to do a proper controlled scientific investigation, but nevertheless the case result that got diagnosed and responded well to treatment, were very, very promising!

NOTE: All treatment studies for effect of antibiotic treatment have been done only on patients with negative direct test for Borrelia ... so of course there is little if any benefit from antibiotic treatment in these trials!

If we really want to evaluate results of antibiotic treatment for CURRENTLY ACTIVE Borrelia infection, we must be 100% certain that the patient really HAS A CURRENTLY ACTIVE BORRELIA INFECTION!
... we must be able to detect the Borrelia (antigen) infection reliably with direct optimized methods (diary, microscopy, culture,  PCR) before enrolling patients into a treatment study, because enrolling lots of cases with "post-Lyme" persistent symptoms (nerve damage) with negative antigen test will of course  skew the results towards NO CERTAIN EFFECT in proportion with the group that does no longer have ACTIVITY CYCLE (weekly, monthly)- previous studies like Klempners is pseudoscience, is not useful to anybody, rather is very harmful to those really infected, that are denied treament that can help them, really!   
  
Since the project (stopped after 50 enrolled patients in 2007, for more reasons) I found a few more cases with ringforms in RBCs, including the latest, ON43, described above ...


UPDATED INFORMATION
IS NECESSARY TO INCREASE OUR DOCTORS AWARENESS - therefore I write this little "review" ...
Probably tick bitten (long term) sick patient, that do not regain their health quickly after conventional treatment for Borrelia infection, should NO LONGER be dismissed as malingerers or psychosomatic cases, but should be investigated thoroughly and be properly investigated as "best science" show us is possible for both Borrelia culture in BSK-H (PCR subtyping possible after, rarely possible on fresh patient sample due to very low amount of spirochaetes catched in the sample), Line Western blot serology analysis (can detect many different antibodies, including strain specific) plus be properly evaluated for the many other tickborne co-infections, that we now know that ticks may harbour and can infect humans and pets and farm animals with (how about eating relatively raw meat as has become modern?), using the new types of PCR tests described in the articles below, that are able to detect all subtypes of tickborne patogens, and using locally found variant strains in IFA ... until we know more about what we have, and can develop more specific serology test methods!
Any laboratory worker (any doctor really) should learn and be able to do buffy-coat smears and stain blood with Giemsa and examine in the microscope, therefore I explain how and why in this article! 

Babesia venatorum (EU1)


PubMed search for Babesi*+(EU1+OR+venatorum):  http://www.ncbi.nlm.nih.gov/pubmed?term=Babesi*+(EU1+OR+venatorum)

Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe. (2003)
http://wwwnc.cdc.gov/eid/article/9/8/02-0748_article.htm
"Subsequent testing of serum specimens from both [Babesia EU1] patients showed IFA reactivity to B. divergens but not to B. microti antigens; serum from the Italian patient was also tested for reactivity to WA1 antigens and was negative."

Patient characteristics table:
(CDC unfortunately had no luck in producing infection in jirds by the experimental inoculation of patients blood, so the IFA was done with B. divergens as test  antigen!)
The italian patient: "Titers of 1:64 (specimen from October 28, 1998) and 1:256 (February 15, 1999) in testing at both CDC and the Clinical Institute of Hygiene of the University of Vienna"
The Austrian patient: "Titers of 1:256 (July 31, 2000) and 1:1,024 (August 8, 2000) in testing at CDC and titers of 1:64 (July 31) and 1:1,000 (August 8) in testing at the Clinical Institute of Hygiene of the University of Vienna"


For comparison: The danish patients titer to unknown test antigen was 1:125 ...

[Babesiosis in an immunocompromised German patient]. (tysk, 2008)
http://www.ncbi.nlm.nih.gov/pubmed/18270666

"Babesiosis was confirmed by polymerase chain reaction (PCR) and the parasite was identified as EU1. Serology was negative. Therapy with clindamycin and quinine induced remission. Following a relapse, retreatment with atovaquone and azithromycin was initiated. After several months, seroconversion occurred and the patient cleared the parasite 8 months after first admission."


Transport of Babesia venatorum-infected Ixodes ricinus to Norway by northward migrating passerine birds. (2011):
http://www.ncbi.nlm.nih.gov/pubmed/21699719    http://www.actavetscand.com/content/pdf/1751-0147-53-41.pdf  (I had problem with the version saved in PMC!)
METHODS: Passerine birds were examined for ticks at four bird observatories along the southern Norwegian coast during the spring migrations of 2003, 2004 and 2005. The presence of Babesia was detected in the nymphs of Ixodes ricinus by real-time PCR. Positive samples were confirmed using PCR, cloning and phylogenetic analyses.
RESULTS: Of 512 ticks examined, real-time PCR revealed five to be positive (1.0%). Of these, four generated products that indicated the presence of Babesia spp.; each of these were confirmed to be from Babesia venatorum (EU1). Two of the four B. venatorum-positive ticks were caught from birds having an eastern migratory route (P< 0.001).
[hence the other two may have come from a southern route, perhaps they flew over Denmark?]
CONCLUSIONS: Birds transport millions of ticks across the North Sea, the Skagerrak and the Kattegat every year. Thus, even with the low prevalence of Babesia-infected ticks, a substantial number of infected ticks will be transported into Norway each year. Therefore, there is a continuous risk for introduction of new Babesia spp. into areas where I. ricinus can survive.

Therefore, let us look at the incidence of Babesia spp. detected in ticks, humans or animals in other European countries, located south of Denmark, from where the passerine birds may have come and flying over Denmark on their way to Norway, fouraging, resting underways  ... leaving ticks or perhaps aquiring new ticks underways?

Ticks and associated pathogens collected from domestic animals in the Netherlands. (2007) (=6 mentioned above)
http://www.ncbi.nlm.nih.gov/pubmed/17979540
"Following an outbreak of autochthonous canine babesiosis in the Netherlands, a request made to veterinarians and the public to collect ticks from companion animals resulted in 4298 ticks submitted between July 2005 and October 2006 to our center.
Ticks were identified as Ixodes ricinus adults (2907/4298, 67.6%), Ixodes sp. nymphs (529/4298, 12.3%) and Ixodes sp. larvae (385/4298, 9.0%), I. hexagonus adults (328/4298, 7.6%), Dermacentor reticulatus (72/4298, 1.7%), and several other exotic tick species such as Amblyomma flavomaculatum (formerly Aponomma flavomaculatum), Hyalomma marginatum rufipes, Rhipicephalus sanguineus, and R. turanicus (55/4298, 1.3%). Eight localities were surveyed for the presence of local D. reticulatus, a tick not indigenous to the Netherlands, based on multiple submissions of D. reticulatus ticks from these areas. D. reticulatus was collected from the vegetation in six of these localities, confirming the presence of populations of this tick in the Netherlands.
Adult I. ricinus (n=251), I. hexagonus (n=237), and D. reticulatus (n=344) ticks were selected at random and subsequently screened by polymerase chain reaction (PCR) and reverse line blot (RLB) hybridization for the presence of Borrelia, Babesia, Theileria, Anaplasma, Ehrlichia, and Rickettsia species.
I. ricinus ticks were infected with Rickettsia helvetica (24.7%), spirochetes belonging to the Borrelia burgdorferi sensu lato group (7.2%), the Ehrlichia-like "Schotii" variant (2.4%) [Anaplasmataceae PDF 2004,  suggested nomenclature for "Schottii variant" is ‘Candidatus Neoehrlichia mikurensis’ acc. to fig. 2], Anaplasma phagocytophilum (1.6%), Babesia sp. (EU1) (1.2%), Babesia divergens (0.4%), and Babesia microti (0.4%). [EU1:divergens/microti 3,5:1]
A. phagocytophilum (5.9%) and R. helvetica (0.8%) were also detected in adult I. hexagonus ticks. Spotted fever group Rickettsiae, previously reported as Rickettsia sp. DnS14/RpA4 (14.0%), and Borrelia burgdorferi sensu lato (0.3%) were detected in the D. reticulatus ticks, which appeared to be free from B. canis infection. We concluded that a much broader spectrum of ticks and tick-borne pathogens is present in the Netherlands than previously thought, including several potential zoonotic pathogens."

The First Detection of Babesia EU1 and Babesia canis canis in Ixodes ricinus Ticks (Acari, Ixodidae) Collected in Urban and Rural Areas in Northern Poland (2009)
http://www.pjm.microbiology.pl/archive/vol5832009231
"Ixodes ricinus, the most commonly observed tick species in Poland, is a known vector of such pathogenic microorganisms as TBE viruses, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Babesia divergens and B. microti in our country.
Our study aimed to find out whether this tick can also transmit other babesiae of medical and veterinary importance.
DNA extracts of 1392 ticks (314 nymphs, 552 male and 526 female ticks) collected in urban and rural areas in the Pomerania province (northern Poland), were examined by nested PCR for the detection of Babesia spp., using outer primers: 5-22F and 1661R, and inner primers: 455-479F and 793-772R, targeting specific fragment of 18S rRNA gene. Overall, at least 1.6% ticks were found to be infected with babesial parasites. In the case of nymphs, the minimal prevalence was 0.6%, and it was approx. 3-times lower than in adults (1.9%). Percentages of infected males and females were comparable (2.0% vs. 1.7%). Sequences of 15/22 PCR-derived fragments of 18S rRNA gene demonstrated 100% similarities with the sequence of Babesia EU1 (proposed name B. venatorum) (acc. no. AY046575) (n=13) and with B. canis canis (acc. no. AY321119) (n = 2), deposited in the GenBank database. The partial 18S rDNA sequences of Babesia EU1 and B. c. canis obtained by us from I. ricinus have been deposited in GenBank, accession nos. GQ325619 and GQ325620, respectively. The results obtained suggest the possible role of I. ricinus as a source of microorganisms, which have been identified as agents of human and canine babesiosis, respectively, in Europe. To our knowledge this is the first report on the occurrence of Babesia EU1 and B. c. canis in I. ricinus in Poland."

Babesia sp. EU1 infection in a forest reindeer, The Netherlands. (2011)
http://www.ncbi.nlm.nih.gov/pubmed/21529420    http://wwwnc.cdc.gov/eid/article/17/5/10-1834_article.htm
"All blood and tissue samples from each organ tested were positive only for Babesia sp. EU1. ... 
Of I. ricinus ticks from the Netherlands, ≈1% are infected with Babesia sp. EU1 (6). The only confirmed reservoir host of Babesia sp. EU1 is roe deer (Capreolus capreolus) (9). The infected forest reindeer resided in a zoo in an area without direct contact with roe deer, although roe deer are abundant in the forests surrounding the zoo."  


Co-infection with Borrelia species and other tick-borne pathogens in humans: two cases from
Poland. (2010)
http://www.ncbi.nlm.nih.gov/pubmed/21186774     http://www.aaem.pl/pdf/17309.pdf
"The co-infection rate of these pathogens was found to be rather low (Borrelia spp./Anaplasma phagocytophilum – 4.2%, 1/24; Borrelia spp./Babesia spp. – 4.2%, 1/24). However, due to the increased prevalence of Borrelia spp. in Ixodes ricinus ticks in Poland and the recent emergence of new tick-borne infections, it is necessary to carefully evaluate the true risk of human infection with several pathogens using more sensitive and reliable diagnostic tools.
This is the first report of human infection with Babesia spp. in Poland that has been confirmed by molecular techniques with homology of 98.9% to B. divergens or Babesia EU1."


Excerpts:
"Three recently described human cases identified in Italy, Austria and Germany were caused by infection with a species named EU1, exhibiting molecular characteristics distinct
from those of B. divergens [16, 18]. The rodent species B. microti is also present throughout Europe, although there has only been one verified case of human babesiosis due to infection with this species [20]. In recent years, co-infections of humans acquired from Ixodes ricinus ticks have been observed quite frequently in the United States [1, 4, 38] and in Europe [24, 44]. However, only three such cases have been reported so far in Poland [12, 17, 27].
In this retrospective study we examined Borrelia-seropositive individuals from southeastern Poland, where I. ricinus ticks are highly endemic, for coinfection with A. phagocytophilum and Babesia species."
"Examination of a smear of the Babesia-positive blood sample stained with Giemsa revealed Babesia spp. infection with a very low level parasitemia of 0.02% (Fig. 3)." ...

"Human co-infections with the pathogens Borrelia spp. and A. phagocytophilum with clinically (erythema migrans) and serologically (seroconversion) confirmed Lyme borreliosis as well as asymptomatic anaplasmosis (positive serology or PCR for A. phagocytophilum) have been described in Poland [12, 17] and other countries [23, 25]. Nevertheless, the few previous reports of such co-infections indicate that the resulting disease is more severe and prolonged 
[39*]. Since the late 1950s, two species of Babesia, B. divergens from cattle in Europe and B. microti from rodents in the northeastern and upper midwestern parts of the USA, have been shown to infect humans [26]. The species B. microti, B. divergens, B. odocoilei-like, and Babesia EU1 are known to be prevalent in I. ricinus ticks across Europe, including Poland [9, 15, 33, 43]."  [no clinical case description]

[39*] = Coinfections acquired from ixodes ticks. 2006 PDF  ..
NB In this review I found more wrong statements that has been disproven, so read carefully and check all statements, that may now be out of date!
For instance the authors claim that ONLY Bbss, B. afzelii and B. garinii are known to be human pathogenic species.
However, cases of other Borrelia burdorferi sensu lato had already been detected and reported before publication of the review:

Borrelia bisettii (Strle F, Slovenia 1999 PM "Thus far four Borrelia species were found by isolation to cause disease in humans: B. afzelii, B. garinii, B. burgdorferi sensu stricto, and B. bissettii.")

B. lusitaniae, Portugal 2004 PDF    Borrelia valaisiana Greece, 2004 HTM   Borrelia spielmanii (so far only detected in early Lyme disease AFAIK?, i.e. in EM rash) 2005 PDF
- so the authors were clearly not fully up-to-date with the published literature (or were ignorant?) when they wrote that statement in 2006! 

Proving causation is not possible after 10 years of chronic illness, as in the cases with B. lusitaniae and B. valaisiana, however both these patients were described as clinically compatible with what some of us (ILADS stance) call "chronic Lyme disease", while other colleagues (of IDSA stance) stubbornly deny the existence of persistent borrelia infection in "chronic Lyme disease", where they usually mean  "typical NEURO-borreliosis" (ass. w/ B. garinii infection), despite there are lots of culture, PCR and/or microscopy verified LATE and POST-TREATMENT cases that has been described since 1989; because the IDSA chronic Lyme denialists usually do not reference any of the published works, that speak against their stance (ignorance seem to fluorish among them), I made a list of references -
persistent-borreliosis - and put it on my website in 2003, to make it easier for myself and others to check up on which references are MISSING / not discussed in articles authored by folks of IDSA stance ;)

A more recent review over Lyme borrelia diversity is discussed by Stanek (EUCALB, usually stick close to the IDSA stance in US) in "The expanding Lyme Borrelia complex—clinical significance of genomic species?" (1911) PDF ..  he still claim that only the 3 old wellknown Borrelia genospecies known from 1992 are with certainty humane pathogenic, about the others he describe them as "single" or "a few case reports", like on page 491, he writes "the presence of B. valaisiana [in neuroborreliosis] was confirmed in three cases [1]."
So I wonder, HOW many cases does it take for them to accept that Borrelia infection sometimes can be a chronic persistent perhaps life-long infection, especially in some susceptible individuals?

The recently published NIH-primate study by Embers et al. (Full text + PDF) demonstrate long term persistence of Borrelia despite 3 months ceftriaxone treatment (error in drug was corrected), this has not yet been added to my persistent borreliosis reference list!
"Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4–6 months later.* Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals.
B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered postdissemination, in a primate host."


many humans have been sick for much longer, over a year to several years, before getting diagnosed and treated first time for their Borrelia infection; esp. those with B. afzelii here in Europa are diagnosed late, because focus is set only on detecting "typical" neuroborreliosis [Bannwarth syndrome] with high spinal-Borrelia-antibody index etc. - which is characteristic for B. garinii neuroborreliosis, but not in B. afzelii neuroborreliosis (Strle CID 2006 PDF)

As long as genotyping it not yet generally avaiable to us for diagnosis we will never know with certainty what type of Borrelia(s) the patient got infected with!
We need access to culture for Borrelia (at least in cases that do not get well on standard tretment for Borrelia) as well as co-infections in cases suspect of chronic late or persistent Lyme borreliose, where antigen specific diagnosis of Borrelia will support necessity for treatment and aid in choice of treatment.

MY VIEWPOINT IS:
Up 
until now so far 18 named genospecies have been placed in the Borrelia burgdorferi sensu lato (Bbsl) spp. complex, and so far 8 of these have been detected in material from human Borreliosis cases from Europe since 2004! - the enlarging spectrum show us that other strains of borrelia than the 3 old one, may eventually turn out be humane pathogenic, could perhaps not be detected before the PCR era, because these strains are so antigenically different, that the commonly used commercial Borrelia antibody tests, that are based on antigens from the 3 old types of Borrelia burgdorferi sensu lato, may not detect antibodies directed against the new types; more of the cases with other types have shown to been seronegative or very low seropositive, despite they harbour Borrelia!
Previously detectable Lyme antibodies have been shown to disappear, despite culture verified persistence of Borrelia in ligamentous tissue (Häupl et al. and more) ...

During very active Lyme borreliosis with detectable spirochetes by simple microscopy, many patients may test seronegative in same plasma fraction, which can be explained by immunecomplex bound antibodies are not being measured: http://kroun.ulmarweb.dk/Borrelia-IC.html (in danish, but reference mentioned are in english) ... some of them will "seroconvert" 1-2 month into clinically effective antibiotic treatment, when there is no longer antigens in blood as often (shift to monthly relapse cycle); when treatment is stopped some of them relapses again, and again spirochetes can be detected, and they may turn seronegative .. retreatment usually works and they may "seroconvert" again, when the cycle shift to monthly  .. UNDERSTANDING THE DYNAMICS OF TEST RESULTS IN RELAPSING INFECTION IS A NECESSITY! 

Because IDSA base the very diagnosis of Lyme borreliosis primarily on that the patient MUST HAVE POSITIVE SEROLOGY, two-tier, because they have chosen to use the CDC EPIDEMIC SURVEILLANCE CRITERIA as DIAGNOSTIC CRITERIA for Lyme borreliosis in the USA, despite CDC actually have warned against it! - this BLOCK for openminded discussion on the many cases of proven persistent and othen seronegative borreliosis cases. They have chosen to define"Lyme disease" in a certain way, not accepting a broader perspective; most of the controversies arises from the fact that some of the lead authors in IDSA on Borrelia have personal conflicting economic interest in serology tests!

From my viewpoint I talk about "Borrelia infection" in a much broader sence, i.e. the patient is infected, but not necessarily clinically ill, when ANY borrelia spp., broad defined i.e. including TBRF and LBRF sub-spp., can be detected by direct test methods like culture, PCR and/or microscopy (plus the patient has history indicative of borrelia like known tickbites, EM rashes, previously positive serology test) ...
Borrelia infection (antigen present and able to revert and cause symptoms, but is currently an inactive infection) may be latent, asymptomatic for weeks, months to even many years, just like in tertiary syfilis, there may be a potential risk of later relapse, in case of immune depression; such have been described in persistent borreliosis list, there are lots of published reports that supports this, really!

The KEY POINT in diagnosis of ACTIVE INFECTION is EASY, SIMPLE namely the RE-OCCURRENCE OF THE CYCLICAL CLINICAL RELAPSE PATTERN, WHICH ALLOWS US TO DETECT SPIROCHETES IN BLOOD BY MICROSCOPY DURING THE FIRST DAY OF EVERY ACTIVE FLARE PERIOD! .. in blood taken at other times spirochetes are usually not detectable!
If the patient has a "weekly" relapse pattern spirochetes can be detected in 1 per 10 days of the cycle (10% chance of lucky catch, while the risk of missing the catch is 90%); in patients on a monthly cycle 1 per 30 days, the chance of lucky catch is 3%, while the chance of missing the catch is 97%) if the clinician does NOT time the sampling for direct detection to happen day one of a new flare!
The antigen in my project cases reacted with added Borrelia antibody that had been absorbed for TBRF species and tested by the producer to be specific for Borrelia burgdorferi sensu lato (KPL).

First when we know which strain the patients have got and can compare to clinical status, it will become possible to evaluate the clinical significance of the different strains of Lyme borreliae!

Let us spare the discussion now and agree that we do not yet have information enough to make a definite conclusion on this issue, that would serve the sick best to keep an open mind to what may be possible, I think!
- there may be strains out there that has not even been found yet ...

The authors [of 39] also claim that larvae of Ixodes is not transovarially infected with Borrelia. However I know more studies proving that if the mother tick is systemically infected with Borrelia (which from 5-30% have been found to be) the Borrelia infection will be transmitted to 100% of eggs and the progeny larvae will be born infected (for instance old works by Burgdorfer himself, I have not made a list yet, is on my to-do-list)! ... More PCR studies on unfed Ixodes larvae have shown that 5-10% may harbour Borrelia spp. and therefore is potentially contagious!

There may be more disproven out-of-date or skewed statements in articles depending on "stance" of the authors, so read articles with great caution! .. be very thorough and check reference-list, not only if they reference correctly, but also which articles are missing, that they should have referenced and discussed in their context, really!

Occurrence of
Babesia spp., Rickettsia spp. and Bartonella spp. in Ixodes ricinus in Bavarian public parks, Germany.
http://www.ncbi.nlm.nih.gov/pubmed/21762494     http://www.parasitesandvectors.com/content/pdf/1756-3305-4-135.pdf
"The following prevalences were detected:
Babesia
spp.: 0.4% (n = 17, including one pool of two larvae) in 2009 and 0.5 to 0.7% (n = 11, including one pool of five larvae) in 2010;
Rickettsia spp.: 6.4 to 7.7% (n = 285, including 16 pools of 76 larvae) in 2009.
DNA of Bartonella spp. in I. ricinus in Bavarian public parks could not be identified.
Sequence analysis revealed the following species: Babesia sp. EU1 (n = 25), B. divergens (n = 1), B. divergens/capreoli (n = 1), B. gibsoni-like (n = 1), R. helvetica (n = 272), R. monacensis IrR/Munich (n = 12) and unspecified R. monacensis (n = 1).
The majority of coinfections were R. helvetica with A. phagocytophilum (n = 27), but coinfections between Babesia spp. and A. phagocytophilum, or Babesia spp. and R. helvetica were also detected."


=> Ratio of Babesia venatorum (EU1):Babesia divergens found in the Bavarian park ticks was 25:1 
i.e. humans and pets visiting that city park is at 25 times relative higher risk of getting infection with Babesia EU1 compared to Babesia divergens, that is, if they get a Babesia infection, since less than 1% of the examined ticks were found infected with Babesia species the general risk of aquiring Babesia infection is still quite low!

Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland. (2006)
http://www.ncbi.nlm.nih.gov/pubmed/16841874    http://www.aaem.pl/pdf/13065.pdf
"The serological assays presently available are not able to discriminate between EU1 and B. divergens infections [14], hence all the cases reported to date have been ascribed to B. divergens."
"We have described a new PCR method based on the 18S rRNA gene that allows the detection of Babesia in I. ricinus ticks and in blood samples. The amplified fragments show an important number of mutations. In addition, the variability of the target fragments (25.3%) is similar to the variability of the complete gene sequences (20.3%). Because of this variability, the short amplicon of 411-452 bp of the 18S rRNA gene may be used for accurate species resolution. Indeed, we could differentiate all the Babesia species we considered (Fig. 2). Moreover, in the B. canis cluster, we clearly could separate the two subspecies B. canis vogeli and B. canis canis (Fig. 2). The analytical sensitivity of the PCR assays has been shown to be 27.5 fg/reaction of B. divergens DNA. However, the majority of the preview reports describing PCR for the detection of Babesia in ticks did not report the sensitivity, with the exception of a study which showed a limit of detection of 50 organisms/ml of canine
whole blood [3]. ...
This is the first study reporting the presence of Babesia sp. EU1 in Switzerland. This organism was detected in ticks collected in two areas located North (Neuchātel) and South of the Alps (Ticino). The presence in Switzerland of B. microti and B. divergens was also confirmed. ...
Recently, Meer-Scherrer et al. [20] described the firsthuman case due to B. microti in a Swiss patient. However, the behaviour and the virulence of the European B. microti strains might be different from those of North America. Interestingly, in another study, it has been suggested that B. microti in the United States may be more virulent for humans than the B. microti-like isolated in Japan [25]. It is important to consider that B. microti actually consists of a genetical species complex based on the 18S rRNA gene: Babesia isolated in rodents divide in two clades, one zoonotic, including our samples, and the other maintained only in rodents [11].
Recently, two cases of human babesiosis, in Italy and Austria, have been attributed to EU1 [14]. Moreover, in Slovenia ticks have been found infected by this species [6]. The EU1 sequences obtained in these different countries, including Switzerland, are identical or very close (99.8% of similarity) and thus no specific geographic association may be identified. An important issue concerning Babesia sp. EU1 is to determine whether it is an emergent species."

First molecular evidence of potentially zoonotic Babesia microti and Babesia sp. EU1 in Ixodes ricinus ticks in Belgium. (2011)
http://www.ncbi.nlm.nih.gov/pubmed/20575647    
"We report the first molecular evidence of the presence of Babesia sp. EU1 and Babesia microti in Ixodes ricinus ticks in Belgium. A 1-year national survey collected 1005 ticks from cats and dogs. A polymerase chain reaction technique amplifying a part of the 18S rRNA gene detected Babesia spp. in 11 out of 841 selected and validated tick extracts.
Subsequent sequencing identified Ba. microti (n=3) and Babesia sp. EU1 (n=6). This study has demonstrated a low infection rate (1.31% with 95% CI: 0.65-2.33) of Babesia spp. carriage in I. ricinus ticks in Belgium but, for the first time, reports two potentially zoonotic species belonging to this genus. Coinfection with Ba. microti and Borrelia burgdorferi sensu stricto also was demonstrated. In addition, this study clearly demonstrates that inhibitors of polymerase chain reaction amplification are present in engorged ticks.
"

Questing ticks in suburban forest are infected by at least six tick-borne pathogens. [France, 2011]:
http://www.ncbi.nlm.nih.gov/pubmed/21158500
"...  Pathogen prevalence rates were evaluated by polymerase chain reaction detection and sequencing in questing ticks, individually for adults and in pools of 10 for nymphs. In addition to finding micro-organisms corresponding to symbionts, we found high prevalence rates of B. burgdorferi s.l. (32% of adult females and 10% of nymphs)low to moderate ones of Anaplasma phagocytophilum (~1%), spotted fever group Rickettsia spp. (~6%), Babesia sp. EU1 (~1%), Bartonella birtlesii (0.1%), and Francisella tularensis (!1%).
Our findings extend the knowledge of the geographical distribution of these endemic and emergent pathogens and support the conclusion that ticks are important vectors of pathogenic micro-organisms in suburban forests. Moreover, tick coinfection with multiple pathogens was found to occur frequently, which poses a serious challenge for diagnosis and appropriate treatment. The incrimination of these pathogens in potentially severe pathologies requires widespread surveillance to assess the risk of infection, thereby facilitating diagnosis and treatment, as well as raising local awareness of tick-borne diseases."
and

Detection and characterization of Babesia species in Ixodes ticks in Estonia. (2011)
http://www.ncbi.nlm.nih.gov/pubmed/21395407
"The presence of Babesia spp. was studied in 2603 Ixodes ricinus and Ixodes persulcatus ticks collected at seven sites in Estonia. By reverse line blot screening, spp. was detected in 36 (1.4%) ticks, among them 18 (0.7%) were further recognized by a BabesiaBabesia microti probe, 3 (0.1%) by a Babesia divergens probe, and the other 15 (0.6%) were recognized only by the universal Babesia spp. "catch all" probe. Sequence analyses of 6 of these 15 samples revealed that all of them belonged to Babesia sp. EU1. B. microti was detected in both tick species I. ricinus and I. persulcatus at the seven sites, whereas B. divergens-like and Babesia sp. EU1Babesia sp. EU1 share a high rate of similarity and are closely related to sequences from other European countries, Siberia, and United States. The present study demonstrated for the first time the existence and distribution of Babesia spp. in I. persulcatus and I. ricinus ticks in Estonia."

Prevalence of three zoonotic Babesia species in Ixodes ricinus (Linné, 1758) nymphs in a suburban forest in Switzerland. (2011)
http://www.ncbi.nlm.nih.gov/pubmed/21395425
"... The aim here was to determine how frequently these [Babesia] species infect I. ricinus nymphs in a suburban forest and to determine their prevalence over 3 years along a pathway delimited in four different sections.
Babesia spp. was detected and identified in 44/2568 (1.7%) I. ricinus nymphs using Reverse Line Blot.
B. venatorum [EU1] was infecting 1.1% (27/2568) of nymphs, B. divergens 0.2% (4/2568), and B. microti 0.7% (13/1908).
Tick infection rates by these three Babesia species between years were not different except for B. microti, which was significantly less frequent in ticks in 2008 than in 2006 and 2007 according to a test using trusted intervals of percentages. B. microti was displaying the greater difference of prevalence among sampling sections, ranging from 1.6% in section 1 to 0% in section 4. The presence of these three Babesia species that are of medical relevance in a suburban forest where I. ricinus tick density is high requires attention from physicians, particularly for patients presenting unspecific symptoms and for patients who are immunocompromised, and who have history of contact with tick biotopes."

Pathogens of emerging tick-borne diseases, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp., in Ixodes ticks collected from rodents at four sites in Switzerland (Canton of Bern). (2011)
http://www.ncbi.nlm.nih.gov/pubmed/21417929
"The prevalence of Babesia spp. reached 2.4% and identification at the species level revealed B. venatorum (1.7%) and B. microti (0.4%)."

Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy. (2012)
http://www.ncbi.nlm.nih.gov/pubmed/22452970      http://www.parasitesandvectors.com/content/pdf/1756-3305-5-61.pdf
"Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. [N= Neoehrlichia] mikurensis" (10.5%).
Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%).
Co-infections by more than one pathogen were diagnosed in 22% of infected ticks.

Antibody prevalence and molecular identification of babesia spp. In roe deer in France. (2012)
http://www.ncbi.nlm.nih.gov/pubmed/22493116    
"In a region-wide serologic study carried out in 2004 on free-ranging hunted roe deer in various landscapes, we found that 58% of the animals (237 out of 406) were antibody positive for Babesia divergens antigen. Serologic and infection status was also analyzed for 327 roe deer live-trapped in two fenced forest areas over 5 yr (2004-08). For two consecutive years during this period, 92 and 94% of the deer in these closed populations were antibody-positive for B. divergens.
Babesia
spp. were isolated in autologous red blood cell culture for 131 of the trapped animals (40%).
Molecular typing was done on 76 isolates with polymerase chain reaction (PCR)-restriction fragment length polymorphism methods targeted at the 18S ribosomal subunit gene (18 isolates) and the Bd37 gene coding for a merozoļte surface antigen implicated in a protective response (60 isolates).
Results indicated continuous cocirculation of B. capreoli and B. venatorum in both forests and possible coinfection of animals with both species. No infection with B. divergens was detected. Fifteen isolates were confirmed to be B. capreoli by sequencing part of the 18S rRNA gene. Using PCR detection of the Bd37 gene, all nine isolates of B. venatorum in this study were negative, whereas the 15 confirmed and 50 putative B. capreoli isolates showed very variable restriction profiles, distinct from those known for Bd37 in B. divergens. Two isolates showed conflicting results, suggestive of mixed infection."


Babesia duncani (WA1)

PubMed Babesi*+(WA1+OR+duncan*): http://www.ncbi.nlm.nih.gov/pubmed?term=Babesi*+(WA1+OR+duncan*)

This strain is described as Babesia-gibsoni-like and is known to cross react in serology test with Babesia gibsoni, while patients with Babesia WA1 usually test negative on antibody test for B. microti!

- above ref. from the German city park, described finding 1 Babesia gibsoni-like! ... perhaps it is closely related to be called WA1-like, since WA1 is also B. gibsoni-like?

I need mention this because sign of WA1 infection has been described in Europe ONCE, and in Denmark of all places:

1996 danish conf. abstract: 14 of 132 (10,6%) of danish neuroborreliosis patients and 2 of 50 donors tested seropositive for WA1 antibodies:
"Two of the patients with elevated titers to WA1 had a prolonged disease course, one with elevated liver enzymes."

The third described case of transfusion-transmitted Babesia duncani. (2011).
http://www.ncbi.nlm.nih.gov/pubmed/22168221    
Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024).
The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index
WA1 parasite isolated in 1991.
The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA.
The implicated donor - a 67-year-old California resident - had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008.

THIS IS SO INSTRUCTIVE that I do not find it necessary to list other reports that likewise show that
commonly used assays for B. microti does not detect infection with B. duncani (WA1)


It shows the timing, long persistence of babesia if not treated, risk for blood recipient if the asymptomatic low level carrier decides to donate blood. 
There are no screening for Babesia, blood banks just ask if the patient feel healthy, do not exclude previous borrelia / tickbitten as donors, do not apply any tests for Babesia on donors blood. 
The Japanese donor transmitted case - http://lymerick.net/York2004/Japanese-donor-Babesia.htm    http://www.ncbi.nlm.nih.gov/pubmed?term=babesi*+Japan+donor -
http://jcm.asm.org/content/38/12/4511.full.pdf
"The donor's serum exhibited a high antibody titer against the isolate from the patient, whereas it exhibited only a weak cross-reaction against B. microti strains isolated in the United States."

Indirect fluorescent-antibody test (IFAT). An approximately 50% suspension of infected RBCs which had 30 to 50% parasitemia was prepared in phosphatebuffered
saline (PBS; pH 7.2) containing 50% fetal bovine serum. Roughly 0.3-ml  aliquots were spread into each well of a 24-well HT Coating Slide (MS 342 BL; Bokusui Brown, Tokyo, Japan) and were then dried. The slides were fixed in acetone for 5 min and were then immediately transferred into PBS to lyse the RBCs. Following removal of solution by briefly blotting with a filter paper, the slides were placed in a moisturized chamber, and 15 ml of serial twofold dilutions of serum specimens, starting from 1:25, was added to each well. After 1 h of incubation at room temperature, the slides were washed in PBS, and 15 ml of fluorescein isothiocyanate-labeled protein A (EY Laboratories, Inc., San Mateo, Calif.) diluted 1:200 in 5% fetal bovine serum–PBS was added to each well. The slides were incubated at room temperature for 1 h and washed in PBS. Component A of the Slowfade antifade kit (Molecular Probes, Eugene, Oreg.) was mounted onto each well, and cover glasses were placed on the slides. Fluorescent parasites in RBCs were observed with a fluorescence microscope at a magnification of 3200.

Reference strains of B. microti. The Gray strain (4) was a gift from J. Dickerson, Division of Parasitic Diseases, Centers for Disease Control and Prevention.
Strain Gray-Mo, a mouse-adapted substrain of the Gray strain, has been described previously (15). The GI and AJ strains were provided by H. Saeki, Nippon Veterinary and Animal Science University. Syrian hamsters were used for propagation of the Gray strain, and the antibodies in their convalescentphase sera were used as the specific antibodies. The Gray-Mo, GI and AJ strains were propagated in C.B-17 scid mice, and antisera against these strains were prepared with BALB/c mice.

- shows another problem, that there may be local variants in different places of the world, where strains of genetically similar strains, differ enough from each other  in surface antigens, to not be readily detected by a B. microti anitbody test based on a strain from the USA!
=> test should be done with local microbe strains! 

Babesia divergens–like Infection, Washington State (2004)
http://wwwnc.cdc.gov/eid/article/10/4/03-0377_article.htm
Most reported U.S. zoonotic cases of babesiosis have occurred in the Northeast and been caused by Babesia microti.
In Washington State, three cases of babesiosis have been reported previously, which were caused by WA1 (for “Washington 1”)-type parasites.
We investigated a case of babesiosis in Washington in an 82–year-old man whose spleen had been removed and whose parasitemia level was 41.4%.
The complete 18S ribosomal RNA gene of the parasite was amplified from specimens of his whole blood by polymerase chain reaction.
Phylogenetic analysis showed the parasite is most closely related, but not identical, to B. divergens (similarity score, 99.5%), a bovine parasite in Europe.
By indirect fluorescent-antibody testing, his serum reacted to B. divergens but not to B. microti or WA1 antigens.
This case demonstrates that babesiosis can be caused by novel parasites detectable by manual examination of blood smears but not by serologic or molecular testing for B. microti or WA1-type parasites.


THE KEY POINT!  ...  
Neg. antibody test and negative PCR test can not be used to rule out Babesia infections, particularly NOT when ringforms can be detected by simple  microscopy, which still remains "the gold standard" test! 
Negative thin and thick blood smears can not be used to rule o

Malaria:

Wikipedia om Malaria detection methods: http://en.wikipedia.org/wiki/Malaria_antigen_detection_tests

QBC Quantitative buffy-coat method:

PubMed QBC+Malaria:
http://www.ncbi.nlm.nih.gov/pubmed?term=QBC+Malaria

QBC Malaria: http://www.malariasite.com/malaria/QBC.htm

QBC Malaria vivax QBC Malaria vivax QBC Malaria vivax
QBC Malaria falciparum QBC Malaria falciparum QBC Malaria falciparum
Note: The QBC is as screen method fast and more sensitive, but if positive by QBC normal thin and thick blood smear examinations should also be done, for optimal strain identification

List of references on the use of QBC Malaria method:
http://www.qbcdiagnostics.com/products/fm/malaria/studies.asp

Application and Evaluation of QBC Malaria Diagnosis in a Holoendemic Area. (1994)
http://www.ncbi.nlm.nih.gov/pubmed/7812314
"QBC proved more sensitive than the thick-film method, detecting - on day 14 of the in vivo test - low parasitaemias that had gone undetected by thick film. Lastly, this study reports on the conversion of QBC readings (parasitaemia per field) into thick-field terms (number of parasites per microliter of blood), with the aim of measuring the degree of recurring parasitaemia."

Diagnosis of Malaria by Acridine Orange Fluorescent Microscopy in an Endemic Area of Venezuela (1996)
http://www.bioline.org.br/pdf?oc96013x

Detection of Plasmodia in Acridine Orange Stained Capillary Tubes (The QBC System). (1990)
http://www.ncbi.nlm.nih.gov/pubmed/2098913

Malaria Diagnosis. A brief review. (2009)
http://www.ncbi.nlm.nih.gov/pubmed/19488414   http://www.parasitol.or.kr/kjp/Synapse/Data/PDFData/0066KJP/kjp-47-93.pdf
"Nowadays, portable fluorescent microscopes usinglight emitting diode (LED) technology, and pre-prepared glass slides with fluorescent reagent to label parasites, are available commercially [38]. Although the QBC technique is simple, reliable, and user-friendly, it requires specialized instrumentation, is more costly than conventional light microscopy, and is poor at determining species and numbers of parasites."

38: Partec reagents and accessories.  http://www.partec.com
  http://www.partec.com/reagents-accessories/essential-healthcare/reagent-kits.html
05-8951 Partec Rapid Malaria Test, 200 tests
Ready-prepared and ready-to-use Malaria test slides with dried-in reagents for easy-to-perform 1-step protocol and immediate analysis. Maximum shelf life: 12 months.


Evaluation of the QBC Method to Detect Malaria Infections in Field Surveys. (1994)
http://www.ncbi.nlm.nih.gov/pubmed/7925060
"We conclude that the QBC is quicker, with high sensitivity, and will prove useful in clinical and epidemiological screening, especially when parasitaemia is low."

Direct acridine orange fluorescence examination of blood slides compared to current techniques for malaria diagnosis. (1996)
http://www.tropicalmedandhygienejrnl.net/article/S0035-9203%2896%2990300-4/abstract
"The renewed interest in the use of fluorochromes for malaria diagnosis prompted us to evaluate the acridine orange fluorescence technique on blood slides, and to compare it with established techniques using thick and thin blood films and the QBC(TM) malaria test, using the Giemsa-stained thick film technique as our standard method for comparison.
We compared 123 positively diagnosed cases and 120 negative cases.
For primary samples (day 0), the sensitivity of the thin blood film fluorescence acridine orange technique (AO) was 96·4%, and its specificity was 95·1%.
In cases of imported malaria, with a prevalence rate of 16·2%, the positive predictive value was 79·2% and the negative predictive value 99·3%.
Sensitivity of AO was significantly higher than that of Giemsa-stained thin blood films for parasitaemias <5000/μL. The potential of AO for species diagnosis of Plasmodium was 85·2%, using Giemsa-stained thin films as the reference technique.
Where QBC(TM) imposes a cost limitation, especially in developing countries, despite its high performance, the AO diagnostic technique is a valuable alternative, because of its simplicity, almost negligible cost, and its diagnostic reliability. The method may also have potential value in the diagnosis of other microbiological diseases.

Feasibility and limitations of acridine orange fluorescence technique using a Malaria Diagnosis Microscope in Myanmar.
http://www.ncbi.nlm.nih.gov/pubmed/12530504    http://www.lib.okayama-u.ac.jp/www/acta/pdf/56_5_219.pdf
About: "Acridine Orange Technique. AOTF were stained by placing a small amount of AO solution in a single strip down the center of the cover slip (18 x 18 or 24 x 24 mm) that was laid on a filter paper or a paper towel. Holding the thick blood-smeared slide at both ends with the smear facing downwards, the technician pressed the slide gently against the cover slip with AO stain. Any excess stain squeezed out was adsorbed by the underlying filter paper or paper towel. The blood smear was examined immediately under an MDM-ESL microscope starting from low magnification 200 X, where parasites could be easily spotted. The parasites were best identified under 600 X magnification."

(preliminary .. there may be more interesting articles .. check back later)